High-Content Imaging in Daphnia magna : From Mechanistic Ecotoxicology to Effect-Based Water Quality Assessment
Year: 2026
Type: dissertation
Abstract: Biodiversity is declining globally, and chemical pollution represents a potentially underestimated pressure on ecosystems. Chemical hazard assessment relies on standardized toxicity tests that focus on apical endpoints and evaluate chemicals individually. Because environmental exposures involve complex mixtures and toxic effects arise through molecular and cellular mechanisms, these approaches may overlook important sublethal and mixture-related effects. New approach methodologies (NAMs) can strengthen chemical hazard assessment. This thesis aimed to develop, to my knowledge, the first high-content imaging method for detecting multiple biological effects in the aquatic model organism Daphnia magna. A whole-organism workflow was optimized using the molecular probes JC-1 and BioTracker blue as indicators of mitochondrial membrane potential, Calcein AM as an indicator of esterase activity and cellular viability, and Rhodamine B as an indicator of multixenobiotic resistance (MXR)-associated transport. Deep learning-based image analysis enabled quantitative and spatially resolved assessment of toxicological effects. In Paper I, JC-1 staining was optimized, demonstrating that inappropriate concentrations reduced assay sensitivity or generated nonspecific fluorescence signals. The study also established 5% ethanol as a suitable anaesthetic for temporarily immobilizing D. magna during imaging. In Paper II, Calcein AM staining was optimized, and feeding conditions were shown to affect staining quality. Neonates not previously exposed to food were therefore most suitable for imaging. In both studies, sublethal effects of environmental contaminants were detected below concentrations causing immobilization in OECD TG 202 and, for some chemicals, at environmentally relevant concentrations. Responses were also detected earlier than apical effects and may predict mortality after longer exposures. In Paper III, Rhodamine B, Biotracker blue and Calcein AM were combined for multiplexed high-content imaging. D. magna were exposed to cyclosporin A, carbonyl cyanide 3-chlorophenylhydrazone, and triphenyl phosphate as positive controls. Each probe responded most strongly to its corresponding model compound, while effects were also detected across other endpoints, illustrating the complex nature of cellular toxicity. Defined ternary mixtures produced distinct multiparametric toxicity profiles. Effects linked to all three model compounds were detected, including at lower concentrations than when the compounds were tested individually. In Paper IV, the developed method identified distinct toxicity profiles in extracts from four rivers in the Dhaka region of Bangladesh. Effects occurred at environmentally relevant concentrations, in some samples below the field-equivalent concentration. Integration with chemical profiles supported interpretation of site-specific toxicity and potential pollution sources, demonstrating the method’s utility for effect-based assessment of complex environmental mixtures. Overall, multiplexed high-content imaging rapidly generated sensitive, quantitative, spatially resolved and mechanistically informative data on multiple sublethal responses in intact D. magna. By combining several biological endpoints in a single automated workflow, the method provides a promising complementary NAM for mechanistic ecotoxicology, chemical mixture assessment and effect-based characterization of complex environmental samples.
Source: DiVA (Stockholm University)
Author: Abele, Cedric
Language: English
Subfield: Health, Toxicology and Mutagenesis
Field: Environmental Science
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7214409753